Gottumukkala Venkateswara Rao et al. / Journal of Pharmacy Research 2012,5(1),165-168 Research Article Available online through ISSN: 0974-6943 http://jprsolutions.info Alkamides and their biological activity from longum Linn. Gottumukkala Venkateswara Rao*, Kolisetty Sambasiva Rao#,Triptikumar Mukhopadhyay, M. S. L. Madhavi CavinKare Research Centre, 12, Poonamalle Road, Ekkattuthangal,Chennai-600 032, Tamil Nadu, India # Dabur India Ltd., Plot No. 22, Site. IV, Sahibabad 201010, Ghaziabad (UP), India Received on:20-09-2011; Revised on: 15-10-2011; Accepted on:10-12-2011

ABSTRACT From the air dried fruits of Piper longum Linn., four secondary metabolites: brachystamide D (1), isopiperlongumine (2), piperine (3) and piplartine (4) were isolated. Their structures were elucidated on the basis of extensive physical and chemical analysis (UV, IR, 1H and 13C NMR) and comparison with known compounds. One of the compound, isopiperlongumine (2) is new to the literature and brachystamide D (1) is the first report from this . The methanolic extract, its fractions and isolated compounds were examined for in-vitro dendrite elongation inhibition property and showed good activity.

Key words: Piper longum, amides, secondary metabolites, dendrite elongation inhibition. INTRODUCTION Piper longum (long pepper) belongs to family and about 30 Based on our continuing interest on the isolation of bioactive secondary species have been recorded in India from this family. Piper longum is an metabolites from for personal care applications,[8-15] we under took a aromatic climber with perennial woody roots occurring in the hotter parts of chemical examination of the fruits of Piper longum. We report here the India, from Central Himialayas to Assam and evergreen forests of western isolation and structure elucidation of four compounds 1-4, of which one was ghats from Konkan to Travencore. It is being cultivated on a large scale in found to be new to the literature and another compound was isolated for the lime soil, near Chirapunji region, Assam. The fruits are used as spice and also first time from this plant. Structure of the compounds were established based in pickles and preserves. The fruits as well as the roots are attributed with on their physical and spectral data and also comparison with those reported numerous medicinal uses, and are also used for diseases of respiratory tract, in the literature. The present paper describes the isolation of chemical con- viz., cough, bronchitis, asthma, etc., as counter-irritant and analgesic when stituents from active fraction and its dendrite elongation inhibition studies. applied locally for muscular pain and inflammation; as snuff in coma and drowsiness and internally as carminative; as sedative in insomnia and epi- MATERIALS AND METHODS lepsy; as general tonic and heamatinic; as cholagogue in obstruction of bile duct and gall bladder, in dysentery and leprosy; as an emmnenagogue and General abortifacient; as anthelmintic.[1] The alcoholic extracts of the dry fruits and Melting point: Hot plate (Tempo) and are not corrected; IR: Prestige 21 FT aqueous extracts of the leaves showed activity against Micrococus pyogenes IR (Shimadzu); NMR: 1H, 13C NMR (Bruker AMX 400); Mass spectrum: var. aureus and E. coli. [1] The ether extract of the fruits showed larvicidal Jeol SX 102/DA 600 mass spectrometer. Column chromatography (CC) was properties.[1] In Chota Nagpur, the root is used to ferment rice beer. In carried on a silica gel column (100-200 mesh). Purity of the samples was [2] Andaman Islands, the leaves are chewed like leaves. The fruits have checked by TLC on pre-coated aluminum sheets, silica gel 60 F254 (20 X 20 a pungent pepper like taste and produce salvation and numbness of the cm, 0.2mm thickness, Merck) and compounds were detected under UV light mouth. The fruits of the P. longum has shown the presence of the alkaloids, (254 & 366 nm) and spraying with 5% sulphuric acid in methanol followed piperine, piplartine and an unidentified alkaloid. An unidentified alkaloid has by heating the plates at 110oC for 5 min. The chemical shift values are been found to produce salvation, numbness and a tingling sensation of mu- reported in ppm (d) units and the coupling constants (J) are in Hz. cous membrane of the mouth. The same unidentified alkaloid also showed Plant material significant in vitro anti-tubercular activity against Mycobacterium tubercu- The seeds of Piper longum (1.0kg) were collected from local market in Chennai, losis H-37 Rv strain; it inhibited the growth of the bacillus in 20mg/ml con- Tamil Nadu (India) in July 2008 and identified by Dr. P. Santhan, Plant centration.[1] The 50 % ethanolic, benzene and acetone extracts of P. longum Taxonomist, Durva Herbal Centre, Chennai, India. A voucher specimen of showed anti-fertility activity. [3] The compound, piperine isolated from P. this plant was deposited in Cavinkare Research Centre, Chennai, India. longum showed central nervous depressant activity. [4] The alcoholic extract of the fruits of the plant, P. longum and its component, piperine shows Extraction and Isolation procedure immuno-modulatory and anti-tumor promoting activity. [5] The compound, The seeds of Piper longum (309 g) were exhaustively extracted with metha- piperine was also reported to have anti-platelet effect.[6] The fruit extract of nol (2.0 L) by using soxhlet apparatus. The solvent was removed by rotary P. longum and four of its constituents showed antifungal properties.[7] evaporator under reduced pressure at ~ 40oC and 33.5 g crude extract was obtained. The crude methanol extract showed good dendrite elongation activ- *Corresponding author. ity and melanin inhibition property. The methanolic extract (25g) was Gottumukkala Venkateswara Rao adsorbed on silica gel. 250g of silica gel was packed in sintered glass funnel, Principal Scientist on the top adsorbed silica gel was packed and performed vacuum liquid M/s.Cavinkare Research Centre, 12, chromatography using hexane, chloroform, ethyl acetate and methanol (each Poonamalle Road, Ekkattuthangal, 3 L) to get corresponding fractions 3.64g, 10.5g, 3.5g and 2.3g respectively. Chennai 600 032, India All four fractions were submitted for biological activity to identify the active fraction. The chloroform fraction was found to be most active than other fractions.

Journal of Pharmacy Research Vol.5 Issue 1.January 2012 165-168 Gottumukkala Venkateswara Rao et al. / Journal of Pharmacy Research 2012,5(1),165-168 The chloroform concentrate (7.5 g) was chromatographed over silica gel (C-3”, 4”), 28.5 (C-2”), 28.6-29.3 (C-7 to 13), 32.9 (C-6,14), 46.9 (C-1”), column. The column was eluted with hexane: ethyl acetate (7:3, 5.5:4.5 and 100.8 (C-7’), 105.4 (C-5’), 108.2 (C-2’), 120.2 (C-6’), 121.8 (C-2), 128.3 1:1) and 72 fractions (each 35 ml) were collected. Based on the TLC, frac- (C-4), 129.3 (C-15,16), 132.4 (C-1’), 141.3 (C-5), 142.9 (C-3) 145.6 (C-4’), tions showing similar TLC behaviour were combined to obtain six major 147.9 (C-3’), 166.3 (C-1). fractions: Fr.1 (0.35g), Fr. 2 (0.80g), Fr. 3 (0.3g), Fr.4 (1.0g), Fr. 5 (1.5g), Fr. 6 (1.4g). Fr. 1 was found to be lipid, Fraction 2 was showed solid in nature Isopiperlongumine (2; Fig.1). Colorless crystals; mp:156-58oC; UV and crystallized with hexane: chloroform to get colorless crystals [1, (MeOH)nm : 332, 312, 253 ; IR (KBr) cm-1 : 3292 (NH), 1647 (C=O), 1608, [16] 210mg], compound 2 was obtained from fraction 3 by purifying through + 1 1255, 987; EIMS m/z : 273 [M] ; H NMR (400 MHz, CDCl3):? 0.95 (6H, another small column of silica gel followed by re-crystallization with chloro- d, J=6.4 Hz, 3”,4”-H), 1.83 (1H, m, 2”-H), 3.19 (2H, t, J =6.44 Hz, 1”-H), form to get isopiperlongumine as colorless solid [2, 80 mg]. Fraction 4 was 5.92 (1H, d, J=, 14.8Hz, 2-H), 5.99 (2H, s, 7’-H), 6.70 (1H, d, J = 10.7 Hz, concentrated to get residue (1.0 g) which was re-crystallized with chloro- 5-H), 6.89 (1H, dd, J = 10.7, 13.6 Hz, 4-H), 6.90 (1H, d, J = 8.1, Hz, 5’-H), form to get piperine as pale green colored crystals [3, 900 mg, mp: 126- 6.90 (1H, dd, J = 8.1, 1.2 Hz, 6’-H), 7.00 (1H, d, J=1.2 Hz, 2’-H), 7.36 (1H, [17] 13 28°C]. Fraction 5 showed again solid nature after concentration and dd, J = 14.8, 10.6 Hz, 3-H); C NMR (100 MHz, CDCl3,): d 20.0 (C-3”& crystallized with chloroform: ethyl acetate to get colorless crystals [4, 4”), 28.5 (C-2”), 46.8 (C-1”), 101.8 (C-7’), 105.6 (C-5’), 108.4 (C-2’), 282mg].[18, 19] Fraction 6 showed mostly pigments and not pursuit for 122.4 (C-2), 123.1 (C-4), 124.6 (C-5), 124.6 (C-6’), 130.8 (C-1’), 138.8 (C- purification. 3), 140.8 (C-4), 140.9 (C-4’), 148.1 (C-3’), 166.0 (C-1).

o o Brachystamide D (1; Fig.1). Colorless crystals; mp: 88-89 C; UV Piperine (3; Fig. 1). Colorless crystals; mp:126-28 C; UV (CHCl3)nm : (MeOH)nm: 255; IR (KBr) cm-1 : 3302 (NH), 1656 (C=O), 1620 (aromatic), 340; IR (nujol)cm-1 : 1631, 1583, 1490, 1446, 1251, 1029, 997 and 927; + 1 EIMS m/z: 285 [M]+; 1H NMR (400 MHz, CDCl ): d 1.61 (4H, m), 1.68 1253, 1041; EIMS m/z : 425 [M] ; H NMR (400 MHz, CDCl3): d 0.93 (6H, 3 d, J = 6.5 Hz, 3”,4” -H), 1.38-1.49 (14H, m, 7 to 13- H),1.80 (1H, m, 2”-H), (2H, m), 3.59 (4H, s), 5.98 (2H, s), 6.44 (1H, d, J = 14.6 Hz), 6.73 (1H, d, 2.17 (1H, d, J = 2.0 Hz, 6&14-H), 3.18 (2H, m, 1”-H), 5.5 (1H, br s, NH) J = 15.2 Hz), 6.77 (1H, dd, J = 14.5, 8.6 Hz), 6.78 (1H, d, J =8.0 Hz), 6.89 5.75 (1H, d, J=14.9 Hz, 2-H), 5.99 (2H, s,7’-H), 6.00 – 6.20 (3H, m, 4,5,15- (1H, dd, J = 8.0, 1.3 Hz), 6.98 (1H, d, J = 1.2 Hz), 7.41 (1H, dd, J = 14.5, 13 H), 6.28 (1H, d, J=15.7 Hz, 16-H), 6.74 (2H, m, 5’,6’-H), 6.90 (1H, s, 2’-H), 9.7 Hz); C NMR (100 MHz, CDCl3,): d 24.6, 24.6, 25.5, 43.2, 46.7, 101.2, 13 105.6, 108.4, 119.9, 122.4, 125.2, 130.9, 138.1, 142.4, 148.0, 148.1, 165.3. 7.20 (1H, dd, J =14.9, 4.7Hz, 3-H); C NMR (100 MHz, CDCl3): d 20.1

3" o Piplartine (4; Fig. 1). Colorless crystals; mp: 124-26 C UV (CHCl3)nm : -1 2' 14 4 16 12 10 8 6 2 H 2" 322; IR (nujol)cm : 1681, 1620, 1585, 1247, 1132 ,1001 and 813; EIMS m/ O 3' 1 N + 1 z:317 [M] ; H NMR (CDCl3, 400 MHz): d 2.49 (2H, m, 5-H), 4.05 (2H, t, 1' 9 1" 4" 15 13 11 7 3 7' 5 J = 6.6 Hz, 6-H), 3.89 (9H, s, 3 X OCH ’s), 6.05(1H, d, J = 9.6Hz, 3-H), O 3 O 4' 6' 6.81 (2H, s, 2’, 6’-H), 6.96 (1H, dt, J = 9.4, 4.2 Hz, 4-H), 7.43(1H, d, J = 5' 1 13 15.5 Hz, 8’-H), 7.68 (1H, d, J = 15.5 Hz, 7’-H); C NMR (CDCl3, 100 MHz): d 24.7 (C-5), 41.5 (C-6), 56.1 (3’,5’-OMe) 60.8 (4’-OMe), 105.5 (C- 4" 2’& 6’), 121.0 (C-8’), 125.8 (C-3), 125.7 (C-1’), 143.7 (C-7’, 4), 145.3 (C- 5 2' 4 2 H 2" 3’,5’), 153.3 (C-4’), 165.7 (C-2), 168.7 (C-9’). O 3' 1' 1 N 3" 3 1" 7' Dendrite elongation inhibition studies: 6' O O 4' The dendrite elongation inhibition activity of the crude extract and isolated 5' 2 compounds were studied by cell culture method by visual observation of length of dendrites. O

O The melanocyte cells, B16F10 were used for the present study. The cells N H were cultured in DMEM in the presence of 5% carbon dioxide, 10% serum, O Pencillin (100mg/ml), streptomycin (50 mg/ml), Amphotericin B. (2.5mg/ml). 2a 1 X 105 cells were seeded in 60 mm cell culture dish and were incubated with and without the test material for 24 hrs. After 24 hr incubation, the cells were examined under an inverted microscope against negative control.[20] The O method is most precise and reliable. The crude methanol extract and two of 7 5 3 a the isolated compounds were shown good dendrite elongation inhibition O 8 2 b 1 N activity at 50 mg/ml. The dendrite length were recorded in µm and the average 6 4 12 results are tabulated in the table1. a g O 9 11 b 10 3 Table 1: In-Vitro measurement of dendrite length of methanolic extract and isolated compounds, 2 and 3. O 2' 7' 6 H3CO 3' 1 Sl.No. Extract/ Length of the 9' N 5 Compound dendrite in µm 1' 8' 2 6' 4 1 Control (neat cells) 129.3 ± 8.0 H CO 4' O 3 5' 3 2 Crude methanolic extract 80.0 ± 5.7 3 Compound 3 (Piperine) 56.6 ± 4.1 OCH 3 4 4 Compound 2 (Isopiperlongumine) 56.6 ± 5.5

Melanin inhibition activity[21] Figure 1: Compounds from Piper longum The melanin inhibition activity of crude methanolic extract and its fractions

Journal of Pharmacy Research Vol.5 Issue 1.January 2012 165-168 Gottumukkala Venkateswara Rao et al. / Journal of Pharmacy Research 2012,5(1),165-168 were studied in cell lines (B16F10 melanoma cells) along with positive con- second double bond isomerization has been fixed as cis based on the couplings trol, 3-isobutyl-1-methylxanthine (IBMX). The assay method is most reli- constants of C-4 and C-5 protons. Based on the above discussions the able. The crude methanol extract and chloroform fraction showed melanin structure of the compound 2 has been established as isopiperlongumine and inhibition activity at 50 mg/ml whereas ethyl acetate fraction showed moder- is new to literature. ate melanin inhibition activity. The results are tabulated in table 2. Compound 3 was isolated as colorless crystals. Its molecular formula was Table 2: In-Vitro melanin inhibition activity of various fractions determined as C17H19NO3 on the basis of mass spectrum. Its UV spectrum Extract / Fraction % Melanin inhibition showed characteristic absorbance at 340 nm. Its IR spectrum exhibited amide activity at 50 mg/ml carbonyl at 1631 cm-1 and olefinic and aromatic peaks. Its 1H NMR spectrum showed four olefinic proton signals at d 6.44 (1H, d, J = 14.6 Hz), 6.73 (1H, Methanol extract 40 Chloroform fraction 32 d, J = 15.2 Hz), 6.77 (1H, dd, J = 14.5, 8.6 Hz), 7.41 (1H, dd, J = 14.5, 9.7 Ethyl acetate fraction 18 Hz) and three aromatic proton signals d 6.78 (1H, d, J =8.0 Hz), 6.89 (1H, dd, J = 8.0, 1.3 Hz), 6.98 (1H, d, J = 1.2 Hz). Further the spectrum showed RESULT AND DISCUSSION two proton signal at d 5.98 as singlet, three mutliplets at d 1.61 (4H, m), 1.68 o Compound 1 was isolated as colorless crystals, mp=88-89 C. Its molecular (2H, m), 3.59 (4H, s) belongs to piperine moiety. Its carbon spectrum clearly formula was determined as C H NO on the basis of mass spectrum. Its IR 27 39 3 showed a total of 17 carbons of which one carbonyl carbon at d165.3, four -1 spectrum exhibited characteristic broad band at 3302 cm for amine, sharp olefinic carbons at d 119.9, 130.9, 138.1, 142.4 indicates two double bonds -1 -1 band at 1656 cm for amide carbonyl, 1620 cm for aromatic and other in the molecule and six aromatic carbon signals and one methylene dioxy 1 peaks. Its H NMR spectrum showed three aromatic protons at d 6.74 (2H, carbon at d 101.3. Based on the above data, the compound has been identified m), 6.90 (1H, s), six olefinic protons at d 5.75 (1H, d, J=14.9 Hz), 6.00 – 6.20 as piperine whose physical and spectral data, 1H and 13C NMR spectra (3H, m), 6.28 (1H, d, J=15.7 Hz), 7.20 (1H, dd, J =14.9, 4.7Hz) and one agreed very well with piperine isolated earlier from the seeds of Piper methylene dioxy group at d 5.99 (2H, s). Further the spectrum also showed nigrum[23] and Piper longum.[5] one secondary amine signal at d 5.50 as broad singlet and few methylene group signals. Its carbon spectrum clearly showed a total of 27 carbons, of Compound 4 was isolated as colorless crystals, mp=124-26oC. Its molecu- which one amide carbonyl carbon at d 166.3, six olefinic carbons and six lar formula was determined as C17H19NO5. Its UV spectrum showed charac- aromatic carbons. Additionally, the spectrum had shown methylene dioxy teristic absorbance at 322 nm. Its IR spectrum exhibited characteristic bands carbon at d 100.8, ten methylene carbons, one methine carbon and two at 1681cm-1 for unsaturated ketone and 1620 cm-1 for aromatic groups. Its 1H methyl carbons. Based on the above spectral data, the compound was found NMR spectrum showed two methylene signals at d 2.49 (m), 4.05 (t, J = 6.6 1 13 to be amide compound whose H and C NMR spectra agreed well with Hz), three aromatic methoxy signal at d 3.89 as singlet, two a, b-unsatur- those of brachystamide D which was isolated earlier from Piper ated protons at d 6.05 (d, J = 9.6Hz), 6.96 (dt, J = 9.4, 4.2 Hz) Additionally, [16 ] retrofractum. the spectrum also showed two trans olefinic protons at d7.43( d, J = 15.5 Hz), 7.68 (d, J = 15.5 Hz) and two aromatic proton signals at d 6.81 as Compound 2 was isolated as colorless crystals. Its molecular formula was singlet. Its carbon spectrum clearly showed a total of 17 carbons, of which, determined as C H NO based on mass spectrum, M+ 273. Based on the 16 19 3, two amide carbonyl carbon at d 165.7,168.7, four olefinic carbons at d 1 preliminary spectral data, it was recognized as alkamide. Its H NMR showed 121.0, 125.8, 143.7 and 143.7. Further, it showed two methylene carbons at the presence of four olefinic protons at d5.92 (1H, d), 6.70 (1H, d), 6.89 d 24.7, 41.5 and three methoxyl carbons. Based on the above data, the com- (1H, dd), 7.36 (1H, dd). Of which two protons at d 5.92 (1H, d, J=, 14.8Hz), pound has been identified as piplartine whose 1H and 13C NMR spectral data 7.36 (1H, dd, J = 14.8, 10.6 Hz) belongs to a, b- unsaturated carbonyl agreed well with earlier reported compounds from same species.[17] system and are showing large coupling constant, more than 14 Hz indicating [17] that these two protons are in trans position to each other. The other two CONCLUSION olefinic protons are at d 6.70 (1H, d, J = 10.7 Hz, ), 6.89 (1H, dd, J = 10.7, The present study on the fruits of Piper longum resulted in the isolation and 13.6 Hz), indicating that one of the proton coupled with b-proton (J=10.7, characterisation of four compounds 1-4 (Figure 1). The structures of the 14.8) of an a, b-unsaturated proton and other proton coupled with only compound were identified on the basis of spectroscopic data and comparison [22] neighboring proton (J=10.7 Hz). By revealing the coupling constant values, with the literature values. They were found to be brachystamide D (1), the coupled protons are showing a maximum of 10.7 Hz which indicates isopiperlongumine (2), piperine (3) and piplartine (4). The compound, these protons are having cis orientation and trans orientation to other double isopiperlongumine was found to be new to the literature and another compound, bond. The spectrum also showed three aromatic protons at d 6.90 (1H, d, J brachystamide D was isolated for the first time from this plant. The crude = 8.1, Hz), 6.90 (1H, dd, J = 8.1, 1.2 Hz), 7.00 (1H, d, J =1.2Hz), indicates methanolic extract, its fractions and isolated compounds were studied for that the aromatic ring has been substituted with three other groups. Further, dendrite elongation inhibition activity. Compounds 2 and 3 were showed the spectrum showed one methylene dioxy group protons at 5.99 (2H, s,), an good dendrite elongation inhibition property. The methanolic extract and isobutyl group at 0.95 (6H, d, J =6.4 Hz), 1.83 (1H, m), 3.19 (2H, t, J = 6.44 chloroform fractions were showed melanin inhibition activity in B16F10 Hz) which is connected to amide to group. Based on the proton NMR data melanoma cell lines. and comparison with related compounds data, we thought that it may be [22] piperlonguminine type of compound. Its carbon spectrum clearly showed ACKNOWLEDGEMENT a total of 16 carbons, of which, one amide carbonyl carbon at d 166.0, four We thank Mr. C.K. Ranganathan, CMD of CavinKare Pvt.Ltd., Chennai for olefinic carbons at d 122.4, 123.1, 124.6, 138.8, six aromatic carbons, one his interest, constant encouragement and providing necessary facilities. methylene dioxy carbon at d 101.8 and isobutyl group carbons at d 20.0, 20.0, 28.5, 46.8. By comparing the literature, the proton and carbon data of REFERENCES alkamide derivative 2 is found exactly matching with the reported data of 1. Anonymous, The wealth of India- Raw materials, Vol. VIII. CSIR, New piperlonguminine[22] except second double bond stereochemistry. So the Delhi, 1969, p. 96-99. basic skeleton of the compound 2 is similar to that of compound 2a. The 2. Srinivasan KS, The vegetation of Kanyakumari District, Bull Bot Survey, 2, 1960,15.

Journal of Pharmacy Research Vol.5 Issue 1.January 2012 165-168 Gottumukkala Venkateswara Rao et al. / Journal of Pharmacy Research 2012,5(1),165-168 3. Prakash AO, Potentialities of some indigenous plants for antifertility 13. Rao GV, Rao KS, Annamalai T, Radhakrishan N, Mukhopadhyay T, activity Int J Crude Drug Res, 24, 1986, 19-24. Chemical constituents and mushroom tyrosinase inhibition activity 4. Woo EBL, Shin KH, Medicinal plants with anticonvulsant activities, of Chloroxylon swietenia Leaves, Turk J Chem, 33, 2009, 521-26. Soul Taehakkyo Saengyak Yonguso Opjukjip, 18, 1979, 66-70. 14. Rao GV, Radhakrishan N, Mukhopadhyay T, Artoindo-nesianin F, a 5. Sunila ES, Kuttan G, Immunomodulatory and antitumor activity of potent tyrosinase inhibitor from the roots of Artocarpus heterophyllus Piper longum Linn and Piperine, J Ethnopharmacology, 90, 2004, Lam, Ind J Chem, 49B, 2010, 1264-66. 339-46. 15. Rao GV, The chemical constituents and biological studies of Chloroxylon 6. Park BS, Son BJ, Par, YH, Kim TW, Lee SE, Antiplatelet effects of swietenia DC, A review. Indian Drugs, 45, 2008, 5-15. acidamides isolated from the fruits of P. longum Linn, Phytomedicine, 16. Banerji A, Sarkar M, Datta R, Sengupta P, Abraham K, Amides from 14, 2007, 853-55. Piper brachystachyum and Piper retrofractum, Phytochem, 59, 2002, 7. Lee SE, Park BS, Fungicidal activity of pipernonaline, a piperidine 897-901. alkaloid derived from long pepper, Piper longum L., against phyto- 17. Hashimoto K, Yaoi T, Koshiba H, Yoshida T, Maoka T, Fujiwara Y, pathogenic fungi, Crop Protection, 20, 2001, 523-28. Yamamoto Y, Mori K, Phytochemical isomerisation of piperine, a 8. Rao GV, Mukhopadhyay T, Annamalai T, Radhakrishnan N, Sahoo pungent constituent in pepper, Food Sci Technol, 2, 1996, 24-9. MR, Chemical constitutes and biological studies of Origanum vulgare 18. Craig DD, Lee AD, Justin S, Ane W, Deborah KD, Cenocladamide, a Linn, Pharmcog Res, 3, 2011, 143-45 dihydropyridone alkaloid from Piper cenocladum, Phytochem, 53, 9. Rao GV, Annamalai T, Mukhopadhay T, Madhavi MSL, Chemical 2000, 51-4. constituents and melanin promotion activity of Cissus quadrangularis 19. Atal CK, Dhar KL, Singh J, The chemistry of Indian Piper species, Linn, Res J Chem Sci, 1, 2011, 25-9. Lloydia, 75, 1975, 256-64. 10. Rao GV, Annamalai T, Sharlene C, Mukhopadhay T, Madhavi MSL, 20. Rao GV, Rao KS, Madhavi MSL, Mukhopadhyay T, Dendrite elonga- Secondary metabolites and biological studies of seeds of Carum carvi tion inhibitor from Piper longum, Indian patent appl. No. 2217, Linn, J Phar Res, 4, 2011, 2126-2128. 2009. 11. Rao GV, Annamalai T, Mukhopadhyay T, Nardal, a new sesquiterpene 21. Lee J, Jung E, Park J, Jung K, Park E, Kim J, Hong S, Park J, Park S, aldehyde from the plant, Nardostachys jatamansi DC, Ind J Chem, Lee S, Park D, Glycyrrhin induces melanogenesis by elevating a cAMP 47B, 2008, 163-65. level in B16 melanoma cells, J Invest Dermatol 124, 2005, 405-11. 12. Rao GV, Rao KS, Annamalai T, Mukhopadhyay T, New coumarin diol 22. Costa SS, Mors WB, Amides of Ottonia corcovadensis, Phytochem, from the plant, Chloroxylon swietenia DC, Ind J Chem, 48B, 2009, 20, 1981, 1305-07. 1041-44. 23. Bina SS, Sabira B, Tahsin , Farhat N, Fatima N, An amides from fruits of Piper nigrum, Phytochem, 45, 1997, 1617-19

Source of support: Nil, Conflict of interest: None Declared

Journal of Pharmacy Research Vol.5 Issue 1.January 2012 165-168